Suppression subtractive hybridization (SSH) technique was used to screen up-regulated genes in regenerating liver. The cDNA from rat regenerating liver tissue was used as the tester, and that from normal liver was used as the driver. After cloning, a subtraction cDNA library of 900 clones was obtained. These 900 clones were further analyzed by differential gene expression screening, and 50 clones with obvious overexpression in regenerating liver were identified. Sequencing analysis and homology searching showed that these clones represent 37 genes, 13 of them are homologous to genes known to be involved in liver regeneration 15 are known genes which are first found to be up-regulated in regenerating liver and 9 are novel genes (ESTs) which have been deposited to GenBank. RNA dot blotting analysis was used to further study the gene expression patterns. The results not only confirmed the up-regulation of these genes in regenerating liver tissue, but also showed that these genes had different expression patterns during the process of liver regeneration. The results implied that these genes might play important roles in liver regeneration.